Targeted removal of the FA2 site on human albumin prevents fatty acid-mediated inhibition of Zn2+ binding.

Zinc is required for virtually all biological processes. In plasma, Zn2+ is predominantly transported by human serum albumin (HSA), which possesses two Zn2+-binding sites of differing affinities (sites A and B). Fatty acids (FAs) are also transported by HSA, with seven structurally characterized FA-binding sites (named FA1-FA7) known. FA binding inhibits Zn2+-HSA interactions, in a manner that can impact upon hemostasis and cellular zinc uptake, but the degree to which binding at specific FA sites contributes to this inhibition is unclear. Wild-type HSA and H9A, H67A, H247A, and Y150F/R257A/S287A (FA2-KO) mutant albumins were expressed in Pichia pastoris. Isothermal titration calorimetry studies revealed that the Zn2+-binding capacity at the high-affinity Zn2+ site (site A) was reduced in H67A and H247A mutants, with site B less affected. The H9A mutation decreased Zn2+ binding at the lower-affinity site, establishing His9 as a site B ligand. Zn2+ binding to HSA and H9A was compromised by palmitate, consistent with FA binding affecting site A. 13C-NMR experiments confirmed that the FA2-KO mutations prohibited FA binding at site FA2. Zn2+ binding to the FA2-KO mutant was unaffected by myristate, suggesting binding at FA2 is solely responsible for inhibition. Molecular dynamics studies identified the steric obstruction exerted by bound FA in site FA2, which impedes the conformational change from open (FA-loaded) to closed (FA-free) states, required for Zn2+ to bind at site A. The successful targeting of the FA2 site will aid functional studies exploring the interplay between circulating FA levels and plasma Zn2+ speciation in health and disease.

Recent Advances in Metalloproteomics.

Interactions between proteins and metal ions and their complexes are important in many areas of the life sciences, including physiology, medicine, and toxicology. Despite the involvement of essential elements in all major processes necessary for sustaining life, metalloproteomes remain ill-defined. This is not only owing to the complexity of metalloproteomes, but also to the non-covalent character of the complexes that most essential metals form, which complicates analysis. Similar issues may also be encountered for some toxic metals. The review discusses recently developed approaches and current challenges for the study of interactions involving entire (sub-)proteomes with such labile metal ions. In the second part, transition metals from the fourth and fifth periods are examined, most of which are xenobiotic and also tend to form more stable and/or inert complexes. A large research area in this respect concerns metallodrug-protein interactions. Particular attention is paid to separation approaches, as these need to be adapted to the reactivity of the metal under consideration.

Juggling cadmium detoxification and zinc homeostasis: A division of labour between the two C. elegans metallothioneins.

The chemical properties of toxic cadmium and essential zinc are very similar, and organisms require intricate mechanisms that drive selective handling of metals. Previously regarded as unspecific "metal sponges", metallothioneins (MTLs) are emerging as metal selectivity filters. By utilizing C. elegans mtl-1 and mtl-2 knockout strains, metal accumulation in single worms, single copy fluorescent-tagged transgenes, isoform specific qPCR and lifespan studies it was possible to demonstrate that the handling of cadmium and zinc by the two C. elegans metallothioneins differs fundamentally: the MTL-2 protein can handle both zinc and cadmium, but when it becomes unavailable, either via a knockout or by elevated cadmium exposure, MTL-1 takes over zinc handling, leaving MTL-2 to sequester cadmium. This division of labour is reflected in the folding behaviour of the proteins: MTL-1 folded well in presence of zinc but not cadmium, the reverse was the case for MTL-2. These differences are in part mediated by a zinc-specific mononuclear His3Cys site in the C-terminal insertion of MTL-1; its removal affected the entire C-terminal domain and may shift its metal selectivity towards zinc. Overall, we uncover how metallothionein isoform-specific responses and protein properties allow C. elegans to differentiate between toxic cadmium and essential zinc.

Structural and biochemical characterisation of Co2+-binding sites on serum albumins and their interplay with fatty acids.

Serum albumin-Co2+ interactions are of clinical importance. They play a role in mediating the physiological effects associated with cobalt toxicity and are central to the albumin cobalt binding (ACB) assay for diagnosis of myocardial ischemia. To further understand these processes, a deeper understanding of albumin-Co2+ interactions is required. Here, we present the first crystallographic structures of human serum albumin (HSA; three structures) and equine serum albumin (ESA; one structure) in complex with Co2+. Amongst a total of sixteen sites bearing a cobalt ion across the structures, two locations were prominent, and they relate to metal-binding sites A and B. Site-directed mutagenesis and isothermal titration calorimetry (ITC) were employed to characterise sites on HSA. The results indicate that His9 and His67 contribute to the primary (putatively corresponding to site B) and secondary Co2+-binding sites (site A), respectively. The presence of additional multiple weak-affinity Co2+ binding sites on HSA was also supported by ITC studies. Furthermore, addition of 5 molar equivalents of the non-esterified fatty acid palmitate (C16:0) reduced the Co2+-binding affinity at both sites A and B. The presence of bound myristate (C14:0) in the HSA crystal structures provided insight into the fatty acid-mediated structural changes that diminish the affinity of the protein toward Co2+. Together, these data provide further support for the idea that ischemia-modified albumin corresponds to albumin with excessive fatty-acid loading. Collectively, our findings provide a comprehensive understanding of the molecular underpinnings governing Co2+ binding to serum albumin.

Bacterial catabolism of membrane phospholipids links marine biogeochemical cycles.

In marine systems, the availability of inorganic phosphate can limit primary production leading to bacterial and phytoplankton utilization of the plethora of organic forms available. Among these are phospholipids that form the lipid bilayer of all cells as well as released extracellular vesicles. However, information on phospholipid degradation is almost nonexistent despite their relevance for biogeochemical cycling. Here, we identify complete catabolic pathways for the degradation of the common phospholipid headgroups phosphocholine (PC) and phosphorylethanolamine (PE) in marine bacteria. Using Phaeobacter sp. MED193 as a model, we provide genetic and biochemical evidence that extracellular hydrolysis of phospholipids liberates the nitrogen-containing substrates ethanolamine and choline. Transporters for ethanolamine (EtoX) and choline (BetT) are ubiquitous and highly expressed in the global ocean throughout the water column, highlighting the importance of phospholipid and especially PE catabolism in situ. Thus, catabolic activation of the ethanolamine and choline degradation pathways, subsequent to phospholipid metabolism, specifically links, and hence unites, the phosphorus, nitrogen, and carbon cycles.

Direct determination of free Zn concentration in samples of biological interest.

The speciation of essential metal ions in biological fluids, such as blood plasma and serum, is of fundamental importance to understand the homeostasis of these elements. The activity of metal ions such as Zn2+ in extracellular media is thought to affect their interaction with membrane-bound transporters, and thus is critical for their cellular uptake. Previous approaches to determine "free" Zn2+ (i.e. the hexa-aquo ion) are based on separation by either chromatography or ultrafiltration, or on metallochromic dyes. However, both types of approach are prone to affect the relevant equilibria. These drawbacks can be circumvented with the electroanalytical technique AGNES (Absence of Gradients and Nernstian Equilibrium Stripping), since it can measure free zinc concentration without perturbing the sample speciation. Here, a Bovine Serum Albumin (BSA) + Zn synthetic mixture and Fetal Bovine Serum (FBS) are analyzed as proof of concept. Adsorption of BSA on the surface of the Hanging Mercury Drop Electrode (HMDE), despite the advantage of its renewal, is so intense that it blocks appropriate attainment of the required equilibrium, and only estimations of [Zn2+] can be derived. In contrast, a rotating disc electrode with a thin mercury film deposited on it (TMF-RDE) is advantageous because of its small volume and enhanced mass transfer. Protein adsorption can be prevented by covering the TMF-RDE with Nafion. A free Zn concentration [Zn2+] = 2.7 nmol L-1 was found at pH 7.0, total Zn 20 µµmol L-1 and BSA 600 µµmol L-1. A sample of FBS with fixed pH 7.2 (MOPS 0.08 mol L-1) yielded [Zn2+] = 0.25 nmol L-1. This methodology opens the way to free metal concentration determinations in biological fluids.

Strategies for Therapeutic Amelioration of Aberrant Plasma Zn2+ Handling in Thrombotic Disease: Targeting Fatty Acid/Serum Albumin-Mediated Effects.

The initiation, maintenance and regulation of blood coagulation is inexorably linked to the actions of Zn2+ in blood plasma. Zn2+ interacts with a variety of haemostatic proteins in the bloodstream including fibrinogen, histidine-rich glycoprotein (HRG) and high molecular weight kininogen (HMWK) to regulate haemostasis. The availability of Zn2+ to bind such proteins is controlled by human serum albumin (HSA), which binds 70-85% of plasma Zn2+ under basal conditions. HSA also binds and transports non-esterified fatty acids (NEFAs). Upon NEFA binding, there is a change in the structure of HSA which leads to a reduction in its affinity for Zn2+. This enables other plasma proteins to better compete for binding of Zn2+. In diseases where elevated plasma NEFA concentrations are a feature, such as obesity and diabetes, there is a concurrent increase in hypercoagulability. Evidence indicates that NEFA-induced perturbation of Zn2+-binding by HSA may contribute to the thrombotic complications frequently observed in these pathophysiological conditions. This review highlights potential interventions, both pharmaceutical and non-pharmaceutical that may be employed to combat this dysregulation. Lifestyle and dietary changes have been shown to reduce plasma NEFA concentrations. Furthermore, drugs that influence NEFA levels such as statins and fibrates may be useful in this context. In severely obese patients, more invasive therapies such as bariatric surgery may be useful. Finally, other potential treatments such as chelation therapies, use of cholesteryl transfer protein (CETP) inhibitors, lipase inhibitors, fatty acid inhibitors and other treatments are highlighted, which with additional research and appropriate clinical trials, could prove useful in the treatment and management of thrombotic disease through amelioration of plasma Zn2+ dysregulation in high-risk individuals.

Albumin-mediated extracellular zinc speciation drives cellular zinc uptake.

The role of the extracellular medium in influencing metal uptake into cells has not been described quantitatively. In a chemically-defined model system containing albumin, zinc influx into endothelial cells correlates with the extracellular free zinc concentration. Allosteric inhibition of zinc-binding to albumin by free fatty acids increased zinc flux.

A single sensor controls large variations in zinc quotas in a marine cyanobacterium.

Marine cyanobacteria are critical players in global nutrient cycles that crucially depend on trace metals in metalloenzymes, including zinc for CO2 fixation and phosphorus acquisition. How strains proliferating in the vast oligotrophic ocean gyres thrive at ultra-low zinc concentrations is currently unknown. Using Synechococcus sp. WH8102 as a model we show that its zinc-sensor protein Zur differs from all other known bacterial Zur proteins in overall structure and the location of its sensory zinc site. Uniquely, Synechococcus Zur activates metallothionein gene expression, which supports cellular zinc quotas spanning two orders of magnitude. Thus, a single zinc sensor facilitates growth across pico- to micromolar zinc concentrations with the bonus of banking this precious resource. The resultant ability to grow well at both ultra-low and excess zinc, together with overall lower zinc requirements, likely contribute to the broad ecological distribution of Synechococcus across the global oceans.

A metallothionein from an open ocean cyanobacterium removes zinc from the sensor protein controlling its transcription.

Bacterial metallothioneins are known for a limited range of phyla including cyanobacteria. We have characterised the BmtA from the marine cyanobacterium Synechococcus sp. WH8102 (SynBmtA). This strain inhabits the open ocean, one of the most nutrient-poor environments on Earth, with very low total and free Zn2+ concentrations. Therefore, the presence of a metallothionein, usually associated with zinc and cadmium tolerance, in this strain is intriguing. Previous transcriptomics work revealed that unprecedentedly, expression of SynBmtA is activated by the Synechococcus sp. WH8102 "zinc uptake regulator" (SynZur) at elevated [Zn2+]. SynBmtA binds four Zn2+ ions, and its first 37 residues adopt the zinc-finger fold characteristic of BmtAs. In contrast, sequence similarity to other BmtAs in the C-terminal stretch is low. This is expected to affect especially the most reactive site in zinc-transfer reactions. Indeed, chelators were unable to extract Zn2+ from SynBmtA, even in the presence of denaturant. This indicates an extremely stable protein fold, with no accessibility to any bound zinc ions in the folded protein. In addition, the zinc-binding affinity of SynBmtA exceeds those of any other metallothioneins. Apo-SynBmtA is capable of removing zinc from the sensory site of SynZur, providing one possible avenue of de-activating transcription of the synbmtA gene. All of these properties are consistent with a role in safely sequestering any excess zinc, to prevent toxic effects. The fact that this strain stores zinc in a metallothionein rather than employing an efflux pump implies that zinc is a valuable resource for Synechococcus sp. WH8102 and related strains.

Fatty acids may influence insulin dynamics through modulation of albumin-Zn2+ interactions.

Insulin is stored within the pancreas in an inactive Zn2+ -bound hexameric form prior to release. Similarly, clinical insulins contain Zn2+ and form multimeric complexes. Upon release from the pancreas or upon injection, insulin only becomes active once Zn2+ disengages from the complex. In plasma and other extracellular fluids, the majority of Zn2+ is bound to human serum albumin (HSA), which plays a vital role in controlling insulin pharmacodynamics by enabling removal of Zn2+ . The Zn2+ -binding properties of HSA are attenuated by non-esterified fatty acids (NEFAs) also transported by HSA. Elevated NEFA concentrations are associated with obesity and type 2 diabetes. Here we present the hypothesis that higher NEFA levels in obese and/or diabetic individuals may contribute to insulin resistance and affect therapeutic insulin dose-response profiles, through modulation of HSA/Zn2+ dynamics. We envisage this novel concept to have important implications for personalized treatments and management of diabetes-related conditions in the future.

Albumin Substitution in Decompensated Liver Cirrhosis: Don't Forget Zinc.

Decompensated liver cirrhosis has a dismal prognosis, with patients surviving on average for 2-4 years after the first diagnosis of ascites. Albumin is an important tool in the therapy of cirrhotic ascites. By virtue of its oncotic properties, it reduces the risk of cardiovascular dysfunction after paracentesis. Treatment with albumin also counteracts the development of hepatorenal syndrome and spontaneous bacterial peritonitis. More recently, the positive impact of long-term albumin supplementation in liver disease, based on its pleiotropic non-oncotic activities, has been recognized. These include transport of endo- and exogenous substances, anti-inflammatory, antioxidant and immunomodulatory activities, and stabilizing effects on the endothelium. Besides the growing recognition that effective albumin therapy requires adjustment of the plasma level to normal physiological values, the search for substances with adjuvant activities is becoming increasingly important. More than 75% of patients with decompensated liver cirrhosis do not only present with hypoalbuminemia but also with zinc deficiency. There is a close relationship between albumin and the essential trace element zinc. First and foremost, albumin is the main carrier of zinc in plasma, and is hence critical for systemic distribution of zinc. In this review, we discuss important functions of albumin in the context of metabolic, immunological, oxidative, transport, and distribution processes, alongside crucial functions and effects of zinc and their mutual dependencies. In particular, we focus on the major role of chronic inflammatory processes in pathogenesis and progression of liver cirrhosis and how albumin therapy and zinc supplementation may affect these processes.

The Interplay between Non-Esterified Fatty Acids and Plasma Zinc and Its Influence on Thrombotic Risk in Obesity and Type 2 Diabetes.

Thrombosis is a major comorbidity of obesity and type-2 diabetes mellitus (T2DM). Despite the development of numerous effective treatments and preventative strategies to address thrombotic disease in such individuals, the incidence of thrombotic complications remains high. This suggests that not all the pathophysiological mechanisms underlying these events have been identified or targeted. Non-esterified fatty acids (NEFAs) are increasingly regarded as a nexus between obesity, insulin resistance, and vascular disease. Notably, plasma NEFA levels are consistently elevated in obesity and T2DM and may impact hemostasis in several ways. A potentially unrecognized route of NEFA-mediated thrombotic activity is their ability to disturb Zn2+ speciation in the plasma. Zn2+ is a potent regulator of coagulation and its availability in the plasma is monitored carefully through buffering by human serum albumin (HSA). The binding of long-chain NEFAs such as palmitate and stearate, however, trigger a conformational change in HSA that reduces its ability to bind Zn2+, thus increasing the ion's availability to bind and activate coagulation proteins. NEFA-mediated perturbation of HSA-Zn2+ binding is thus predicted to contribute to the prothrombotic milieu in obesity and T2DM, representing a novel targetable disease mechanism in these disorders.

Albumin-mediated alteration of plasma zinc speciation by fatty acids modulates blood clotting in type-2 diabetes.

Zn2+ is an essential regulator of coagulation and is released from activated platelets. In plasma, the free Zn2+ concentration is fine-tuned through buffering by human serum albumin (HSA). Importantly, the ability of HSA to bind/buffer Zn2+ is compromised by co-transported non-esterified fatty acids (NEFAs). Given the role of Zn2+ in blood clot formation, we hypothesise that Zn2+ displacement from HSA by NEFAs in certain conditions (such as type 2 diabetes mellitus, T2DM) impacts on the cellular and protein arms of coagulation. To test this hypothesis, we assessed the extent to which increasing concentrations of a range of medium- and long-chain NEFAs reduced Zn2+-binding ability of HSA. Amongst the NEFAs tested, palmitate (16 : 0) and stearate (18 : 0) were the most effective at suppressing zinc-binding, whilst the mono-unsaturated palmitoleate (16 : 1c9) was markedly less effective. Assessment of platelet aggregation and fibrin clotting parameters in purified systems and in pooled plasma suggested that the HSA-mediated impact of the model NEFA myristate on zinc speciation intensified the effects of Zn2+ alone. The effects of elevated Zn2+ alone on fibrin clot density and fibre thickness in a purified protein system were mirrored in samples from T2DM patients, who have derranged NEFA metabolism. Crucially, T2DM individuals had increased total plasma NEFAs compared to controls, with the concentrations of key saturated (myristate, palmitate, stearate) and mono-unsaturated (oleate, cis-vaccenate) NEFAs positively correlating with clot density. Collectively, these data strongly support the concept that elevated NEFA levels contribute to altered coagulation in T2DM through dysregulation of plasma zinc speciation.

A metalloproteomic analysis of interactions between plasma proteins and zinc: elevated fatty acid levels affect zinc distribution.

Serum albumin is a highly abundant plasma protein associated with the transport of metal ions, pharmaceuticals, fatty acids and a variety of small molecules in the blood. Once thought of as a molecular 'sponge', mounting evidence suggests that the albumin-facilitated transport of chemically diverse entities is not independent. One such example is the transport of Zn2+ ions and non-esterified 'free' fatty acids (FFAs) by albumin, both of which bind at high affinity sites located in close proximity. Our previous research suggests that their transport in blood plasma is linked via an allosteric mechanism on serum albumin. In direct competition, albumin-bound FFAs significantly decrease the binding capacity of albumin for Zn2+, with one of the predicted consequences being a change in plasma/serum zinc speciation. Using liquid chromatography (LC), ICP-MS and fluorescence assays, our work provides a quantitative assessment of this phenomenon, and finds that in the presence of high FFA concentrations encountered in various physiological conditions, a significant proportion of albumin-bound Zn2+ is re-distributed amongst plasma/serum proteins. Using peptide mass fingerprinting and immunodetection, we identify candidate acceptor proteins for Zn2+ liberated from albumin. These include histidine-rich glycoprotein (HRG), a multifunctional protein associated with the regulation of blood coagulation, and members of the complement system involved in the innate immune response. Our findings highlight how FFA-mediated changes in extracellular metal speciation might contribute to the progression of certain pathological conditions.

Crosstalk between zinc and free fatty acids in plasma.

In mammalian blood plasma, serum albumin acts as a transport protein for free fatty acids, other lipids and hydrophobic molecules including neurodegenerative peptides, and essential metal ions such as zinc to allow their systemic distribution. Importantly, binding of these chemically extremely diverse entities is not independent, but linked allosterically. One particularly intriguing allosteric link exists between free fatty acid and zinc binding. Albumin thus mediates crosstalk between energy status/metabolism and organismal zinc handling. In recognition of the fact that even small changes in extracellular zinc concentration and speciation modulate the function of many cell types, the albumin-mediated impact of free fatty acid concentration on zinc distribution may be significant for both normal physiological processes including energy metabolism, insulin activity, heparin neutralisation, blood coagulation, and zinc signalling, and a range of disease states, including metabolic syndrome, cardiovascular disease, myocardial ischemia, diabetes, and thrombosis.

Molecular genetic and biochemical characterization of a putative family of zinc metalloproteins in Caenorhabditis elegans.

Four highly similar genes (W08E12.2, W08E12.3, W08E12.4 and W08E12.5) which are consecutively aligned on chromosome IV of the C. elegans genome are predicted to code for small (120-141aa) yet cysteine rich (18-19 cysteines) proteins. Cloning and sequencing of the genomic regions of the isoforms confirmed the presence and order of all genes. The generation of transgenic worms strains with an integrated single copy or extrachromosomal multi-copy PW08E12.3;W08E12.4::GFP uncovered that W08E12.3 and W08E12.4 are constitutively expressed in the pharynx and significantly induced in worms exposed to 100 µM Zn. Knockdown by RNAi did not have a marked consequence on reproductive performance nor was a Zn-dependent effect on nematode growth observed. However, RNAi of these genes led to an accumulation of Zn in the intestinal cells. W08E12.3 was recombinantly expressed in E. coli and the purified protein was shown to be able to bind up to 6.5 Zn molecules at neutral pH. Zn-binding was acid-labile and the apo protein was observed at pH < 4.3. This characterization suggests W08E12.2, W08E12.3, W08E12.4 and W08E12.5 belong to a family of putative Metalloproteins which, akin to metallothioneins, may play an important role in Zn-sensing, homeostasis and/or detoxification.

The type 4 metallothionein from Brassica napus seeds folds in a metal-dependent fashion and favours zinc over other metals.

The problem of handling zinc in the cell is of great importance because zinc is an indispensable micronutrient involved in most physiological processes in all living organisms. Moreover, our understanding of mechanisms governing the discrimination between micronutrients and toxic metals on the level of individual proteins to the whole-organism level is incomplete. Metallothioneins are able to bind heavy metal ions, and roles in zinc homeostasis have been proposed. Here, we have studied the in vitro and in vivo metal-binding abilities of Brassica napus type 4 metallothionein (BnMT4) and its expression in germinating seeds in response to metal treatment. Our studies on the regulation of MT4 expression by metals at early stages of ontogenic development have revealed for the first time that the mRNA levels of BnMT4 were elevated in response to cadmium and zinc. Given this unexpected metalloregulation, and the dramatic differences in protein folding as detected by 1H NMR spectroscopy, we suggest that the BnMT4 protein may not only have a role in zinc homeostasis in early ontogenesis, but also the potential to discriminate between zinc and cadmium, perhaps via differential recognition of Cd- and Zn-complexes by cellular components involved in protein turnover.

Ischemia-modified albumin: Crosstalk between fatty acid and cobalt binding.

Myocardial ischemia is difficult to diagnose effectively with still few well-defined biochemical markers for identification in advance, or in the absence of myocardial necrosis. "Ischemia-modified albumin" (IMA), a form of albumin displaying reduced cobalt-binding affinity, is significantly elevated in ischemic patients, and the albumin cobalt-binding (ACB) assay can measure its level indirectly. Elucidating the molecular mechanism underlying the identity of IMA and the ACB assay hinges on understanding metal-binding properties of albumin. Albumin binds most metal ions and harbours four primary metal binding sites: site A, site B, the N-terminal site (NTS), and the free thiol at Cys34. Previous efforts to clarify the identity of IMA and the causes for its reduced cobalt-binding capacity were focused on the NTS site, but the degree of N-terminal modification could not be correlated to the presence of ischemia. More recent work suggested that Co2+ ions as used in the ACB assay bind preferentially to site B, then to site A, and finally to the NTS. This insight paved the way for a new consistent molecular basis of the ACB assay: albumin is also the main plasma carrier for free fatty acids (FFAs), and binding of a fatty acid to the high-affinity site FA2 results in conformational changes in albumin which prevent metal binding at site A and partially at site B. Thus, this review advances the hypothesis that high IMA levels in myocardial ischemia and many other conditions originate from high plasma FFA levels hampering the binding of Co2+ to sites A and/or B. This is supported by biophysical studies and the co-association of a range of pathological conditions with positive ACB assays and high plasma FFA levels.